title: Mutation rates of TGFBR2 and ACVR2 coding microsatellites in human cells with defective DNA mismatch repair.
abstract: Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFbeta family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1-/-, hMSH6-/-, hMSH3-/-, and MMR-proficient) to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP) gene, allowing a -1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7-35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a -1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2) in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes) and M2 (bright, representing full mutants) were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91 x 10(-4)) and 15 (2.18 x 10(-4)) times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was approximately 3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The -1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background.
authors.affiliation: Department of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Heekyung
authors.initials: H
authors.lastname: Chung
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Young
authors.forename: Claudia G
authors.initials: CG
authors.lastname: Lopez
authors.forename: Thuy-Anh T
authors.initials: TA
authors.lastname: Le
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Lee
authors.forename: Deena
authors.initials: D
authors.lastname: Ream-Robinson
authors.forename: Sherry C
authors.initials: SC
authors.lastname: Huang
authors.forename: John M
authors.initials: JM
authors.lastname: Carethers
mesh_terms: D030301:Activin Receptors, Type II / Q000235:genetics*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D002460:Cell Line; D053843:DNA Mismatch Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005091:Exons; D016368:Frameshift Mutation; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D000070957:MutL Protein Homolog 1; D000074081:MutS Homolog 3 Protein; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C501486:MLH1 protein, human; C078444:MSH3 protein, human; D000074081:MutS Homolog 3 Protein; D009687:Nuclear Proteins; D018125:Receptors, Transforming Growth Factor beta; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D030301:Activin Receptors, Type II; D000077294:Receptor, Transforming Growth Factor-beta Type II; C425585:activin receptor type II-A; D000070957:MutL Protein Homolog 1